The pathway through which viral protein subunits assemble into closed icosahedral shells, capable of packaging DNA molecules, remains obscure. In dsDNA phages the shell assembly process requires coat protein, scaffolding proteins (regulating coat subunit polymerization), and a ring of portal protein to form the channel for DNA packaging at one vertex. In the genetically and biochemically well defined Salmonella phage P22, these processes require about 420 molecules of the gene 5 coat protein, 300 molecules of the gene 8 scaffolding protein, and 12 molecules of the gene 1 portal protein. The differences between the conformation of soluble precursors and their conformations in the mature virion is a major factor complicating the preparation of vaccines using viral subunits and components. Purified precursor forms of the gene 5 coat and gene 8 scaffolding subunits which exhibit the regulated assembly process observed in vivo will be used to identify the complex of coat and scaffolding subunits which initiate icosahedral shell assembly in vitro, and to define the structural intermediates in further growth and closure of procapsid shells. The requirements for the in vitro assembly of the unique DNA packaging portal vertex will be determined by incubating purified precursor portal subunits with coat and scaffolding subunits, to discover the mechanism or cellular factors which insure the assembly of one and only one DNA packaging vertex. Temperature sensitive folding mutations of the thermostable gene 9 tailspike protein identify amino acid residues and sequences directing the polypeptide chain folding and association pathway. The isolation and sequencing of second site mutations which suppress these defects, and their location in the (imminent) crystal structure of the tailspike should identify critical amino acid sequence interactions. The folding pathway for tailspike chain will be probed directly using a bank of monoclonal antibodies directed against the tailspike. Cold sensitive mutations in the tailspike and portal proteins will be analyzed to determine which stages in protein folding or subunit association they interfere with. The precursor portal, scaffolding, and coat structural subunits and precursor shells described above will be isolated in amounts sufficient for growth of crystals suitable for X-ray diffraction.